Specifics of sample preparation of Arthrospira (Spirulina) platensis culture in the study of associated microflora by flow cytometry and scanning electron microscopy
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Abstract
In biotechnological research, it is important to control quantitative characteristics of associated microflora in algal cultures. With the aim of more complete detection and detailed study of associated microflora in Arthrospira (Spirulina) platensis culture, we applied methods of physical and chemical preparation of samples and subsequent investigation of a suspension by flow cytometry after cell staining with SYBR Green I fluorochrome and scanning electron microscopy. As shown, optimal sample preparation options were exposure to a reagent (sodium pyrophosphate or methanol), suspension stirring, ultrasonic treatment, centrifugation, and additional washing procedures. A mean of (27.1 ± 3.9) % of “potential” cumulative bacterial abundance was initially extracted from a cyanobacterial culture. During the following three washes, abundance of microorganisms increased significantly and averaged (88.9 ± 6.3) % (paired t-test; p < 0.05). Further, abundance of microorganisms in the sediment remained insignificant, 6–11%, and could be neglected. As shown, bacterial abundance at different stages of A. platensis cultivation changed from 6.7×106 to 1.7×108 cells·mL−1. The morphological structure of associated microflora in A. platensis at the stationary phase was dominated by large rod-shaped cells (67.2%); the proportion of spiral forms was almost twice as low (30.2%); and spherical forms were even less common (2.6%). The mean bacterial cell volume was (0.16 ± 0.02) μm³, and biomass was 0.022–0.025 g·L−1. The values obtained for indicators of associated microflora are comparable to those provided in literature for A. platensis and other algal cultures. The proposed methods of treatment of A. platensis suspension boosted the efficiency of bacterial separation, facilitated removal of fragments of cyanobacterial trichomes, detritus, and other particles in samples, and provided an opportunity to study associated microflora by flow cytometry and scanning electron microscopy.
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References
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